Detection of calcineurin isoforms

ABSTRACT

A method for detecting isoforms and/or splice variants of Calcineurin or its subunits is established. The inventive method is especially useful for differentiation between the various isoforms of Calcineurin. Herein a polymerase chain reaction of nucleic acids of mammalian tissue is performed and the corresponding products are evaluated. Especially prefered is a reverse transcribed polymerase chain reaction. For the polymerase chain reaction primers for the isoforms and/or splice variants of Calcineurin are used. In a prefered embodiment isoforms and/or splice variants of the catalytic subunit Calcineurin-A are detected.

The invention relates to a method for detection and analysis ofCalcineurin isoforms and/or splice variants thereof and to diagnosticmethods.

Calcineurin (E.C 3.1.3.16) is a serine/threonine phospo-proteinphosphatase and is composed of a catalytic (Calcineurin A) andregulatory (Calcineurin B) subunit. The catalytic subunit is about 60and the regulatory subunit is about 18 kDa. Calcineurin is the onlyprotein phosphatase known to be under the control of calcium andcalmodulin. Binding of calcium and calmodulin is necessary for enzymaticactivity. Calmodulin is bound by the catalytic subunit whereas theregulatory subunit possesses four calcium binding sites.

Calcineurin is discussed in various contextes. For example Calcineurinhas been implicated in neuronal signaling pathways (Klee et al., 1988;Jakel, 1997) but the neuronal function is only poorly understood(Guerini, 1997). Furthermore Calcineurin is discussed in the context ofimmunosuppression, wherein Calcineurin acts via the transcription factorNFAT (Nuclear factor of activated T-cells) on the T-cells response.

Besides these examples it is known, that Calcineurin has been implicatedin a variety of diseases. Thus it has been shown that Calcineurin playsa key role in heart failure (e.g. Force et al., 1999; Izumo and Aoki,1998; Lim and Molkentin, 1999) and stroke (Morioka et al., 1999).Furthermore Calcineurin has been implicated in the pathogenesis ofamyotrophic lateral sclerosis (Ferri et al., 2000), skeletal musclesdiseases (Chin et al., 1998) and epilepsy (Lie et al., 1998).

As mentioned above, Calcineurin is formed by two subunits. Bothsubunits, the catalytic subunit (Calcineurin A) and the regulatorysubunit (Calcineurin B) occur in different isoforms which are encoded bydifferent genes. In mammals, three distinct genes (A-α, A-β, A-γ) forthe catalytic subunit have been characterized, each of which can undergoalternative splicing to yield additional variants. Especially theseisoforms have been implicated in the diseases mentioned above. But up tonow the specific role of the different isoforms in the development andmaintenance of said diseases is not known. Nevertheless, a detection andanalysis of Calcineurin isoforms is a very useful target for diagnosisof different cardiovascular diseases, epilepsy and neurodegenerativediseases, for example. Furthermore a reliable detection and analysismethod for Calcineurin isoforms is a prerequisite for further researchin these fields and other fields. In addition, for example stroke andminor cardiac failures are often unnoticed in the beginning, thereforebiomarkers are also very important to start treatment in time andevaluate the prognosis of the diseases.

Up to now the existing methods for the detection of Calcineurin isoformsare based on immunoblotting techniques. These methods are notwell-suited for research and especially diagnosis, since these methodsare both time-consuming and expensive. Therefore, the invention has theobject to provide a new method for the detection and analysis ofCalcineurin isoforms. By the use of such a method it would be possibleto get insight into the development and maintenance of differentdiseases which are known to be connected with different isoforms ofCalcineurin. Furthermore, the method would provide a useful tool indiagnosis, especially in very early diagnosis of different diseases.

This object is solved by a method according to claim 1. Preferedembodiments of the inventive method are depicted in the dependent claims2 to 10. Primers and a kit suitable for performing the inventive methodare claimed in claims 11 to 17. Claims 18 to 21 refer to the use of saidprimers and claims 22 to 24 relate to a diagnostic method. The wordingof all claims is hereby made to the content of the specification byreference.

In the inventive method for detection and analysis of isoforms and/orsplice variants of calcineurin or its subunits polymerase chainreactions (PCR) are performed. For this step nucleic acids of mammaliantissue are used, special segments are amplified as PCR products andthese products are evaluated. The inventive method is especially usefulfor differentiation between the various isoforms of Calcineurin.

The PCR is preferably a reverse transcribed PCR (RT-PCR). That means,that RNA of the tissue is extracted, especially total RNA. This RNA isreverse transcribed into cDNA by the use of oligo(dT) primers accordingto standard protocols. This cDNA is the template for the following PCRwith specific primers for Calcineurin. The RT-PCR is especiallyprefered, since RNA reflects the actual status of the cell or thetissue, respectively. At the level of DNA all genes for the differentisoforms of Calcineurin are present. It is the transcription of DNA intoRNA, by which it is decided which isoform will be expressed in the cellor the tissue. Furthermore the splicing, which results into differentsplice variants of Calcineurin, takes place at the level of RNA.Therefore, the RNA is of special interest for the inventive method.

After the step of reverse transcription of the RNA into cDNA the PCRwith primers specific for Calcineurin is performed. By this step thedifferent isoforms and/or splice variants of Calcineurin are amplified.

Preferably said primers for the isoforms and/or splice variants used forPCR are not absolutely specific for Calcineurin isoforms of one species,but are essentially specific for Calcineurin isoforms. Said essentiallyspecific primers are usefull in the amplification of Calcineurin ofdifferent species. By the use of these primers the inventive method issuitable for the analysis of Calcineurin of different species likehuman, mouse, rat, dog, cat etc. In an especially prefered embodiment ofthe inventive method the method is used for the analysis of Calcineurinof any mammalian tissue.

Especially prefered are primers for the isoforms and/or splice variantsof the catalytic subunit Calcineurin-A (CN-A). By the use of theseprimers in the inventive manner said isoforms and/or splice variants ofCalcineurin are analyzed. Preferably these isoforms are CN-Aα, CN-Aβand/or CN-Aγ and/or their splice variants. As outlined above it has beenshown that especially these isoforms are of clinical relevance.Nevertheless, the invention comprises the use of primers specific forisoforms and/or splice variants of the regulatory subunit also in orderto analyze these forms of the regulatory subunit.

Advantageously the inventive primers are directed to the 3′-region ofthe relevant sequences, since this region is biologically betteraccessible than the 5′-region. Preferably the primer pairs embraceregions of potential splice variants of the isoforms. It is especiallyprefered that the primers are directed to regions of the isoforms, inwhich the isoforms are especially different.

In a prefered embodiment of the inventive method the following primersfor the isoforms of the catalytic subunit and their splice variants areused.

The primers for CN-Aα are: (SEQ ID NO. 1) atg tgc tca gac gat(CN-Aα-sense 1) gaa ctr gg (SEQ ID NO. 2) ggc atc ctc tcg tta(CN-Aα-antisense att cgg 1).

The primers for CN-Aβ are: (SEQ ID NO. 3) att tgc tct gat gat(CN-Aβ-sense 1) gaa cta at (SEQ ID NO. 4) ggc att ctc tca ttg(CN-Aβ-antisense atc cta 1).

The primers for CN-Aγ are: (SEQ ID NO. 5) cat atg ctc gga tga(CN-Aγ-sense 1) aga aat ga (SEQ ID NO. 2) ggc atc ctc tcg tta(CN-Aγ-antisense att cgg 1).

In the listing of the primers the primers are written in 5′-3′direction. “a” means adenine, “t” means thymine, “g” means guanine and“c” means cytosine. “r” means “a” and “g”. Primers CN-Aα-antisense 1 andCN-Aγ-antisense are identical.

The different isoforms and/or splice variants of calcineurin which arespecific for several diseases as outlined above, are especially presentin nervous tissue. Therefore in a prefered embodiment of the inventivemethod the mammalian tissue as source of nucleic acids is nervoustissue, especially central nervous tissue. Preferably brain tissue isused like amygdala, frontal cortex, hippocampus, piriform cortex, corpusstriatum and/or spinal cord. Especially prefered is the analysis oftissue of both hemispheres of the brain in separate approaches.

In another prefered embodiment of the invention said mammalian tissue iscardiac tissue since Calcineurin isoforms play a key role in heatfailure, for example.

According to the invention the PCR products are evaluated. This could bedone by common methods which are known to an expert skilled in the art.In a prefered embodiment of the inventive method the PCR products areseparated by electrophoresis and visualised by ethidium bromidestaining. Obviously other separating and staining methods are alsopossible. In another embodiment of the inventive method the PCR productsare separated and blotted onto a membrane. These blotted PCR productsare detected by labeled probes, for example by radioactive labeledprobes.

The separation of PCR products is especially prefered in order todifferentiate the isoforms of Calcineurin by differences in themolecular weight.

In another prefered embodiment of the invention the PCR products aredetected without separation of the products. This is performed bytreatment of the solution comprising the PCR products with acorresponding labeled probe and analyzing the interaction of PCRproducts and probe by adequate methods, for example. In this embodimentthe separation step is spared and this is especially advantageous withregard to automation of the whole process.

In a prefered embodiment of the inventive method the evaluation of thePCR products is performed in comparison with an interal standard. Asuited internal standard is always expressed in the same amount and isnot influenced by the diverse conditions of the cell or the tissue. Oneexample of a suited internal standard is actin. According to theinventive method a separate PCR is performed with a primer pair specificfor actin. The resulting PCR product is analysed in parallel to the PCRproducts of Calcineurin and the amounts of the different products arecompared. Thereby it is possible to make quantitative assertionsregarding the expression of the different isoforms and/or splicevariants of Calcineurin.

In a prefered embodiment of the inventive method the resulting PCRproducts are at least partly sequenced. This is especially prefered inorder to identify the PCR products and relate them to certain isoformsand/or splice variants of Calcineurin. Preferably the sequencing of thePCR products is performed by standard protocols known to an expert inthe art.

Moreover the invention comprises primers for PCR with the followingsequences: 1. atg tgc tca gac gat gaa ctr gg (SEQ ID NO. 1) 2. ggc atcctc tcg tta att cgg (SEQ ID NO. 2) 3. att tgc tct gat gat gaa cta at(SEQ ID NO. 3) 4. ggc att ctc tca ttg atc cta (SEQ ID NO. 4) 5. cat atcctc gga tga aga aat ga (SEQ ID NO. 5)

These primers are especially useful for performing PCR in order toobtain the amplification of different isoforms and/or splice variants ofthe catalytic subunit of Calcineurin. By the use of the primers 1. and2. CN-Aα is obtained. By the use of the primers 3. and 4. CN-Aβ isobtained. By the use of the primers 5. and 2. CN-Aγ is obtained.

As template for PCR using these primer pairs generally nucleic acidsextracted from any tissue are suited. Especially prefered are templatesderived from mammalian tissues.

Furthermore the invention comprises a kit for performing a method foranalysis of isoforms and/or splice variants of Calcineurin or itssubunits. The most important ingredients of said kit are primers forisoforms and/or splice variants of a subunit, especially the catalyticsubunit, of Calcineurin. In a prefered embodiment of this inventive kitthe kit comprises different components for performing PCR and/or RT-PCR.Preferably these components are reagents for extraction of RNA and/orprimers for making cDNA and/or primers for an internal standard,especially for actin, and/or reagents for performing PCR, for examplebuffers, salt solutions, nucleotides and water. Regarding the specificprimers for isoforms and/or splice variants of Calcineurin reference ismade to the description above.

Additionally the invention comprises the use of at least one of theprimers above for a method for detecting and analysing isoforms and/orsplice variants of Calcineurin or its subunits. In this embodiment it isespecially prefered that the primer is placed on a DNA chip because suchchips are especially suited for the use in automated processes.Furthermore the invention comprises the use of at least one PCR productobtained from a polymerase chain reaction using at least one of theprimers described above. These PCR products are useful in a method fordetecting and analysing isoforms and/or splice variants of Calcineurinor its subunits. Advantageously the primers and/or PCR products are usedas hybridising probes in order to detect the respective forms ofCalcineurin.

Finally the invention comprises a method for diagnosis of diseasesand/or early recognition of potential diseases, in which Calcineurin isinvolved. Preferably these diseases are cardiovascular diseases,epilepsy and/or neurodegenerative diseases. This inventive method ischaracterized by performing PCR, especially RT-PCR, of nucleic acids ofmammalian tissue and furthermore the evaluation of the PCR productsobtained. Regarding further features of this method reference is made tothe description above.

The described features and further features of the invention result ingreater detail from the following examples, the figures and thesubclaims. The diverse features could be realized alone or incombination with each other.

In the figures it is shown:

FIG. 1: Expression of CN-A isoforms in human brain (human striatum cDNAlibrary). Calcineurin-Aα showed three distinct bands, containing the 341bp and 311 bp splice variants. The Calcineurin-Aβ isoform was onlydetected as a 341 bp splice variant. The calcineurin-Aγ isoform resultedin three different PCR fragments containing the 336 bp and 306 bp splicevariants.

FIG. 2: Expression of calcineurin isoforms in a transgenic mouse modelfor a neurodegenerative disease (amyotrophic lateral sclerosis, ALS).TG: transgenic (ALS), WT: wildtype control (no disease).

FIG. 3: Calcineurin-Aα isoform expression in a model for epilepsy(kindled rats). In the upper panel it is shown the expression inamygdala whereas in the bottom panel insights in the situation inpiriform cortex are given. Both panels reveal typical examples fordifferential expression in kindled animals. Lanes are named as follows:

-   -   kindling: rats were transplanted with electrodes and stimulated    -   electrode: sham operated with an electrode but not stimulated    -   control: sham operated without any transplantation.

FIG. 4: Calcineurin-A isoform expression in a model for cardiovasculardiseases (hypoxic murine brain tissues slices). CN-Aα, CN-Aβ and CN-Aγwere induced in hypoxic murine cortex (upper picture). CN-Aα and CN-Aβwere induced in hypoxic murine hippocampus. CN-Aγ levels are constant inthe range of 20-60 mM 3NPA-treatment (bottom picture).

EXAMPLES

Extraction of RNA was done with Trizol™ reagent (GIBCO, Paisley, USA). 1μg of total RNA was reverse transcribed with oligo(dT)primers accordingto standard protocols. From this reaction, 20-100 ng cDNA was used in aβ-actin PCR for standardisation. The human cDNA was supplied byStratagene (San Diego, USA). The β-actin-primer pairs were supplied byPromega (Heidelberg, Germany). β-actin PCRs were conducted in a 50 μlvolume, containing 5 μl 10×PCR-buffer (500 mM KCl, 100 mM Tris HCl pH9.0, 1% Triton X-100), 3μl 25 mM MgCl₂, 1 μl 10 mM dNTPs, 2.5 μlbeta-actin sense primer and 2.5 μl beta-actin antisense primer, 0.5 to 2μl diluted cDNA (20-100 ng) and RNA free water ad 50 μl.

A “hot start” PCR was done by addition of 2.5 units of Taq polymeraseafter a first heating step at 94° C. for 1 min. The amplificationconditions were 25 cycles of 30 sec. at 94° C. for denaturing, 1 min. at55° C. for annealing and 2 min. at 68° C. for elongation. PCR wasterminated with a 10 min. extension step at 68° C. One fifth of thereactions was analysed by electrophoresis in an ethidium bromide stainedvisigel separation matrix from Stratagene. Quantification of DNA bandswas performed with Multi-Analyst Software from Biorad (München, Germany)by comparing the intensity of Calcineurin with the beta-actin controlcontaining equal amounts of cDNA. For the amplification of Calcineurinisoforms, the primer pairs as described above were used.

Calcineurin PCRs were conducted in a 50 μl volume. They contained 5 μl10×PCR-buffer (500 mM KCl, 100 mM Tris HCl pH 9.0, 1% Triton X-100), 3μl 25 mM MgCl₂, 1 μl 10 mM dNTP-mix, 2.5 μl sense primer, 2.5 μlantisense primer, the calculated amount of diluted cDNA (0.5-100 ng) andRNA free water ad 50 μl. “Hot start” PCR was done by addition of 2.5units of Taq polymerase after a first heating step at 94° C. for 1 min.The primer combinations and amplification conditions were as follows:

-   -   Calcineurin Aα: Primers 1. and 2. with 25 cycles of 30 sec 94°        C., 1 min 60° C. and 2 min 72° C. followed by final extension        step of 10 min 72° C.    -   Calcineurin Aβ: Primers 3. and 4. with 30 cycles of 30 sec 94°        C., 1 min 55° C. and 2 min 72° C. followed by final extension        step of 10 min 72° C.    -   Calcineurin Aγ: Primers 5. and 2. with 30 cycles of 30 sec 94°        C., 1 min 55° C. and 2 min 72° C. followed by final extension        step of 10 min 72° C.

One fifth of the reactions was analysed by electrophoresis in anethidium bromide stained visigel separation matrix (Stratagene).Quantification of band intensity was performed with Multi-AnalystSoftware from Biorad. All calcineurin bands were sequenced to confirmthe specificity of the primer sets.

Mice transgenic for a mutant superoxide dismutase 1 gene associated withfamilial amyotrophic lateral sclerosis were bred. After decapitation andpreparation tissues were immediately frozen in liquid nitrogen. RNA wasprepared from frozen tissue using Trizol™ reagent from GIBCO.

Hypoxic mice and respective controls were treated as described inBüchner et al. (1999), Neuroscience Letters 276: 131-134. Afterdecapitation brain slices were immediately frozen in liquid nitrogen.RNA was prepared according to the protocols of transgenic mice.

Kindled rats were treated and prepared as published in Löscher et al.(1998), Exp. Neurol. 154: 551-559.

The above described methods were used to assay Calcineurin A isoformexpression in three different mammalian species, i.e. mouse, rat andhuman and in six different tissues, i.e. amygdala, frontal cortex,hippocampus, piriform cortex, corpus striatum and spinal cord. As shownin FIG. 2, the bands obtained showed the expected sizes of 341 bp and311 bp for the two Calcineurin-Aα splice variants (all species), 344 bpand 314 bp for the two Calcineurin-Aβ splice variants (rat and mouse;341/311 bp for human), 333 bp and 303 bp for the Calcineurin-Ay splicevariants (mouse and rat; 336/306 bp for human) and 285 bp for theβ-Actin (all species, not shown).

As example of neurodegenerative diseases a model for amyotrophic lateralsclerosis was analysed. Differential expression of Calcineurin wasassayed in mice transgenic for a mutant superoxide dismutase 1 geneassociated with familial amyotrophic lateral sclerosis (FIG. 2). Thetransgenic mice (n=6) showed that at a time point clearly preceeding anypathological changes (45 d) the expression of CN-Aα, CN-Aβ and CN-Aγwere lower than in control animals (n=6). At day 73 (about the time whenthe first pathological changes as degradation of mitochondria orvacuolation of pericarya can be observed), CN-Aα expression is strongerthan in wild types, whereas CN-Aβ and γ expression shows a levelcomparable to wild types. At day 115, when pathological changes can beseen but still before onset of pareses, the CN-Aα expression is slightlylower than controls, whereas CN-Aβ and CN-Aγ mRNA levels were stillcomparable to wild types.

As example for epilepsy kindled rats were analysed. Analyzingcalcineurin isoforms in kindled rats revealed significant differences inthe expression of CN-Aα in the left amygdala and the left part of thepiriform cortex when compared with sham operated controls (transplantedelectrode without stimulation; a typical example is shown in FIG. 3).All together 144 examples were examined, i.e. 6 animals for each braintissue, each hemisphere and each treatment group (kindling, electrodecontrol, untreated control). kindling versus kindling versus electrodeuntreated control kindling versus control kindling versus tissue isoform(significance)* electrode control (%) (significance)* untreated control(%) Amygdala CN-A-α p < 0.01 ″ + 30.59 p < 0.01 ″ + 76.43 (lefthemisphere) 341 bp Amygdala CN-A-α p < 0.01 ″ − 1.80 p < 0.01 ″ + 35.58(left hemisphere) 311 bp Piriform cortex CN-A-α p < 0.01 ″ − 0.83 p <0.01 ″ + 116.2 (left hemisphere) 341 bp Piriform cortex CN-A-α p < 0.01″ + 22.12 p < 0.01 ″ + 264.96 (left hemisphere) 311 bp

As example of cardiovascular diseases hypoxic mice were analyzed. Allcalcineurin-A isoforms were detected in hypoxic mice brain (FIG. 4). AllCalcineurin-A isoforms, i.e. CN-Aα, CN-Aβ and CN-Aγ were induced inhypoxic murine cortex. In hypoxic murine hippocampus only CN-Aα andCN-Aβ were induced, whereas CN-Aγ levels were constant in a range of

1-24. (canceled)
 25. Method for the detecting isoforms or splicevariants of Calcineurin or its subunits, wherein PCR (polymerase chainreaction) of nucleic acids of mammalian tissue is performed, and whereinthe PCR products are evaluated.
 26. The method of claim 25, wherein saidPCR is RT-PCR (reverse transcribed PCR).
 27. The method of claim 25,wherein primers for the isoforms or splice variants are used for PCR.28. The method of claim 27, wherein isoforms or splice variants of thecatalytic subunit Calcineurin-A (CN-A) are detected.
 29. The method ofclaim 28, wherein at least one isoform selected from the groupconsisting of CN-Aα, CN-Aβ, CN-Aγ and their splice variants is detected.30. The method of claim 29, wherein said primers are for CN-Aα (SEQ IDNO. 1): atg tgc tca gac gat gaa ctr gg, and (SEQ ID NO. 2): ggc atc ctctcg tta att cgg, CN-Aβ (SEQ ID NO. 3): att tgc tct gat gat gaa cta at,and (SEQ ID NO. 4): ggc att ctc tca ttg atc cta, CN-Aγ (SEQ ID NO. 5):cat atg ctc gga tga aga aat ga, and (SEQ ID NO. 2): ggc atc ctc tcg ttaatt cgg.


31. The method of claim 25, wherein said mammalian tissue is nervoustissue, central nervous tissue, or brain tissue.
 32. The method of claim25, wherein said mammalian tissue is cardiac tissue.
 33. The method ofclaim 25, wherein the PCR products are separated.
 34. The method ofclaim 25, wherein the PCR products are made visible.
 35. The method ofclaim 34, wherein the PCR products are made visible by staining.
 36. Themethod of claim 25, wherein the evaluation is performed in comparisonwith an internal standard.
 37. The method of claim 36, wherein saidinternal standard is actin.
 38. The method of claim 25, wherein the PCRproducts are at least partly sequenced.
 39. Primer for PCR with thesequence (SEQ ID NO. 1) atg tgc tca gac gat gaa ctr gg.


40. Primer for PCR with the sequence (SEQ ID NO. 2) ggc atc ctc tcg ttaatt cgg.


41. Primer for PCR with the sequence (SEQ ID NO. 3) att tgc tct gat gatgaa cta at.


42. Primer for PCR with the sequence (SEQ ID NO. 4) ggc att ctc tca ttgatc cta.


43. Primer for PCR with the sequence (SEQ ID NO. 5) cat atg ctc gga tgaaga aat ga.


44. Kit for performing a method according to claim 25 comprising primersfor isoforms or splice variants of a subunit of Calcineurin.
 45. The kitof claim 44, further comprising at least one substance selected from thegroup consisting of reagents for extraction of RNA, primers for making cD N A, primers for an internal standard, and reagents for performingPCR.
 46. The kit of claim 44, wherein said subunit of Calcineurin is acatalytic subunit.
 47. The kit of claim 45, wherein said internalstandard is actin.
 48. The kit of claim 44, comprising at least oneprimer selected from the group consisting of a primer with the sequence(SEQ ID NO. 1) atg tgc tca gac gat gaa ctr gg,

a primer with the sequence (SEQ ID NO. 2) ggc atc ctc tcg tta att cgg,

a primer with the sequence (SEQ ID NO. 3) att tgc tct gat gat gaa ctaat,

a primer with the sequence (SEQ ID NO. 4) ggc att ctc tca ttg atc cta,and

a primer with the sequence (SEQ ID NO. 5) cat atg ctc gga tga aga aatga.


49. Method for detecting isoforms or splice variants of Calcineurin orits subunits by the use of at least one primer selected from the groupconsisting of a primer with the sequence (SEQ ID NO. 1) atg tgc tca gacgat gaa ctr gg,

a primer with the sequence (SEQ ID NO. 2) ggc atc ctc tcg tta att cgg,

a primer with the sequence (SEQ ID NO. 3) att tgc tct gat gat gaa ctaat,

a primer with the sequence (SEQ ID NO. 4) ggc att ctc tca ttg atc cta,and

a primer with the sequence (SEQ ID NO. 5) cat atg ctc gga tga aga aatga.


50. The method of claim 49, wherein said primer is placed on a DNA chip.51. Method for detecting isoforms, or splice variants of Calcineurin orits subunits by the use of at least one PCR product obtained from apolymerase chain reaction using at least one primer selected from thegroup consisting of a primer with the sequence (SEQ ID NO. 1) atg tgctca gac gat gaa ctr gg,

a primer with the sequence (SEQ ID NO. 2) ggc atc ctc tcg tta att cgg,

a primer with the sequence (SEQ ID NO. 3) att tgc tct gat gat gaa ctaat,

a primer with the sequence (SEQ ID NO. 4) ggc att ctc tca ttg atc cta,and

a primer with the sequence (SEQ ID NO. 5) cat atg ctc gga tga aga aatga.


52. The method of claim 51, wherein said PCR product is placed on a DNAchip.
 53. Method for diagnosis of diseases or early recognition ofpotential diseases, in which Calcineurin is involved, wherein PCR(polymerase chain reaction) of nucleic acids of mammalian tissue isperformed, and wherein the PCR products are evaluated.
 54. The method ofclaim 53, wherein said PCR is TR-PCR (reverse transcribed PCR).
 55. Themethod of claim 53, wherein said diseases are at least one diseaseselected from the group consisting of cardiovascular diseases, epilepsyand neurodegenerative diseases.
 56. The method of claim 53, whereinprimers for isoforms or splice variants of Calcineurin or its subunitsare used for PCR.
 57. The method of claim 56, wherein isoforms or splicevariants of the catalytic subunit Calcineurin A (CN-A) are detected, andwherein at least one isoform selected from the group consisting ofCN-Aα, CN-Aβ, CN-Aγ and their splice variants is detected.
 58. Themethod of claim 57, wherein said primers are for CN-Aα (SEQ ID NO. 1):atg tgc tca gac gat gaa ctr gg, and (SEQ ID NO. 2): ggc atc ctc tcg ttaatt cgg, CN-Aβ (SEQ ID NO. 3): att tgc tct gat gat gaa cta at, and (SEQID NO. 4): ggc att ctc tca ttg atc cta, CN-Aγ (SEQ ID NO. 5): cat atgctc gga tga aga aat ga, and (SEQ ID NO. 2): ggc atc ctc tcg tta att cgg.